High glucose upregulates connective tissue growth factor expression in human vascular smooth muscle cells

Abstract

Background: Connective tissue growth factor (CTGF) is a potent profibrotic factor, which is implicated in fibroblast proliferation, angiogenesis and extracellular matrix (ECM) synthesis. It is a downstream mediator of some of the effects of transforming growth factor beta (TGFbeta) and is potentially induced by hyperglycemia in human renal mesangial cells. However, whether high glucose could induce the CTGF expression in vascular smooth muscle cells (VSMCs) remains unknown. Therefore, this study was designed to test whether high glucose could regulate CTGF expression in human VSMC. The effect of modulating CTGF expression on VSMC proliferation and migration was further investigated.

Results: Expression of CTGF mRNA was up-regulated as early as 6 hours in cultured human VSMCs after exposed to high glucose condition, followed by ECM components (collagen type I and fibronectin) accumulation. The upregulation of CTGF mRNA appears to be TGFbeta-dependent since anti-TGFbeta antibody blocks the effect of high glucose on CTGF gene expression. A small interference RNA (siRNA) targeting CTGF mRNA (CTGF-siRNA) effectively suppressed CTGF up-regulation stimulated by high glucose up to 79% inhibition. As a consequence of decreased expression of CTGF gene, the deposition of ECM proteins in the VSMC was also declined. Moreover, CTGF-siRNA expressing vector partially inhibited the high glucose-induced VSMC proliferation and migration.

Conclusion: Our data suggest that in the development of macrovascular complications in diabetes, CTGF might be an important factor involved in the patho-physiological responses to high glucose in human VSMCs. In addition, the modulatory effects of CTGF-siRNA during this process suggest that specific targeting CTGF by RNA interference could be useful in preventing intimal hyperplasia in diabetic macrovascular complications.

Figures

Figure 1

High glucose increases CTGF mRNA expression (a) and protein production (b, c) in cultured HUVSMCs. Growth-arrested HUVSMCs were stimulated with high glucose (HG, 25 mmol/L) for different durations. (a) Quantitative RT-PCR (Q-PCR) results. Total cellular RNA was isolated from normal glucose (NG, 5.5 mmol/L), high glucose (HG) or mannitol (25 mmol/L) treated HUVSMCs. After reverse transcription, they were subjected to quantitative PCR (Taqman) analysis to determine CTGF mRNA level. Graph is representative of relative CTGF levels in the various conditions. Experiments were performed five times with the similar results (n = 5 in each group). * P < 0.05 vs NG. (b) Representative Western blot (top) and values of total CTGF production (means ± SEM of 3 experiments, bottom). Results of total CTGF protein production were obtained from densitometric analysis and expressed as ratio of CTGF/β-actin. * P < 0.05 vs NG. (c) Immunocytochemical staining of CTGF protein expression in HUVSMCs (top, magnificent of 400×) and integrated optical density (IOD) of the CTGF staining was measured on the images using the Image-Pro Plus software (bottom). Figure shows a representative experiment out of 3 performed experiments. * P < 0.05 vs scrambled-siRNA transfection under normal glucose media condition. # P < 0.05 vs scrambled-siRNA transfection under high glucose media condition. NG: normal glucose; HG: High glucose; scrambled siRNA: scrambled-siRNA plasmid transfection; siRNA: siRNA-CTGF plasmid transfection.

Figure 2

High glucose-induced CTGF upregulation in HUVSMC is dependent on TGF-β. HUVSMC cells were co-treated with high glucose (HG, 25 mmol/L) and a TGF-β neutralizing antibody (Ab; 10 μg/mL) for 24 hours. (a) Q-PCR results: CTGF mRNA expression was assayed by Q-PCR. Experiments were performed five times with the similar results (n = 5 in each group). * P < 0.05 vs normal glucose (NG). # P < 0.05 vs TGF-β 1. (b) Representative Western blot of 3 performed experiments (top) and values of total CTGF production (mean ± SEM, bottom). * P < 0.05 vs NG. # P < 0.05 vs TGF-β1. NG: normal glucose; HG: high glucose; TGF-β1: TGF-β1 treatment (10 ng/mL); Ab-TGF-β1: TGF-β neutralizing antibody treatment.

Figure 3

siRNA-CTGF transfection reduces basal and high glucose-induced CTGF, collagen type I and FN mRNA (a) and protein expression (b, c and d) in HUVSMC. (a) Q-PCR results: Growth-arrested HUVSMCs were transfected with scrambled or CTGF-siRNA plasmids for 24 hours and then exposed to normal glucose (NG) or high glucose (HG) conditions for 24 to 72 hours. CTGF, collagen type I and FN mRNA expression were assayed by Q-PCR. Experiments were performed five times with the similar results (n = 5 in each group). (b) Representative Western blot (top) and values of total CTGF production (means ± SEM of 3 experiments, bottom). Results of total CTGF protein production were obtained from densitometric analysis and expressed as ratio of CTGF/β-actin. (c) Immunocytochemistry staining of collagen type I protein expression in HUVSMCs (top, magnificent of 400×) and integrated optical density (IOD) of the collagen type I staining was measured on the images using the Image-Pro Plus® software (bottom). Figure shows a representative experiment of 3 performed. (d) Immunocytochemistry staining of fibronectin (FN) protein expression in HUVSMCs (top, magnificent of 400×) and integrated optical density (IOD) of the fibronectin staining was measured on the images using the Image-Pro Plus® software (bottom). Figure shows a representative experiment of 3 performed. * P < 0.05 vs scrambled siRNA transfection under normal glucose (NG) media condition. # P < 0.05 vs scrambled siRNA transfection under high glucose (HG) media condition. Scrambled siRNA: scrambled siRNA plasmid transfection; siRNA: siRNA-CTGF plasmid transfection; NG: normal glucose; HG: High glucose.

Figure 4

CTGF is involved in high glucose-induced proliferation of cultured HUVSMCs. Quiescent cells were transfected with scrambled or CTGF-siRNA expression plasmids for 24 hours and then exposed to HG for 48 hours followed by the assessment of [3H]-thymidine incorporation (a) and cell number counting (b). Each value is the mean ± SEM of 6 separate experiments. * P < 0.05 vs scrambled siRNA transfection under normal glucose (NG) condition. # P < 0.05 vs scrambled siRNA transfection under high glucose (HG) condition. Scrambled siRNA: scrambled siRNA plasmid transfection; siRNA: CTGF-siRNA plasmid transfection.

Figure 5

Role of CTGF in high glucose-induced migration of cultured HUVSMCs. Quiescent cells were transfected with scrambled or CTGF-siRNA expressing plasmid for 24 hours, then exposed to HG for 48 hours, and followed by the measurement of cell migration in a monolayer scratch wound assay. Figure (a) shows a representative result of three mock transfected experiments (Magnification 200×). Figure (b) shows a representative result of three scrambled siRNA plasmid transfected experiments (Magnification 200×). Figure (c) shows a representative result of three CTGF-siRNA plasmid transfected experiments (Magnification 200×). Figure (d) shows the average of migrated cells in three experiments. * P < 0.05 vs mock transfection or scrambled siRNA transfection.

Figure 6

siRNA-CTGF transfection reduces basal and high glucose-induced MMP-2 mRNA (a) and protein expression (b) in HUVSMC. (a) Q-PCR (Taqman) results: Growth-arrested HUVSMCs were transfected with siRNA-CTGF plasmid for 24 hours and then exposed to normal or high glucose conditions for 24 hours. 1 μg of total RNA was reverse-transcribed into cDNA and analyzed for expression of MMP-2 mRNA by real-time PCR. Experiments were performed five times with the similar results (n = 5 in each group). (b) Representative Western blot (top) and values of total CTGF production (means ± SEM of 3 experiments, bottom). Results of total MMP-2 protein production were obtained from densitometric analysis and expressed as ratio CTGF/β-actin. * P < 0.05 vs scrambled siRNA transfection under normal glucose (NG) condition. # P < 0.05 vs scrambled siRNA transfection under high glucose condition (HG). Scrambled siRNA: scrambled siRNA plasmid transfection; siRNA: CTGF-siRNA plasmid transfection.